VAR COD/N 40
The FastDNA SPIN Kit for soil (MP Biomedicals, Irvine, CA) was used to extract the DNA using approximately 0.5 g (wet biomass) from each sample according to the manufacturers instructions. The quantity and quality of the genomic DNA was determined using a NanoDrop spectrophotometer (NanoDrop Technologies) and gel electrophoresis on 1.8% agarose. Extracted genomic DNA was stored at -20C until further use. The universal primers 520f (5-AYTGGGYDTAAAGNG-3) and 802r (5- TACNNGGGTATCTAATCC-3) targeting the V4 region of the 16S rRNA gene for bacteria were used (Claesson et al., 2009). The PCR was performed in a single-step 30 cycle PCR using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) under the following conditions: 94C for 5 min, followed by 30 cycles of 94C for 30 s, 55C for 30 s and 72C for 1.5 min, after which a final elongation step at 72C for 10 min was performed. The expected-size PCR products were purified on 1.8% agarose gel using a commercial kit (ZR Zymoclean Gel DNA Recovery Kit, USA). Adapters and barcordes for Ion Torrent sequencing were included in the primers following a previous strategy (Claesson et al., 2009). Sequencing of the 16S rRNA gene amplicon libraries was carried out using the Ion Torrent Personal Genome Machine (PGM) in the platform at the Biological Research Institute Clemente Estable" (Montevideo, Uruguay).